Project Summary/abstract: HPV associated cancers have increased significantly (>16%) in USA, as per the latest data. Resistance to the current chemotherapy leads to a high recurrence rate and patient death. This resistance has largely been attributed to inter-and intra- tumor heterogeneity. The identification of new therapeutic agents is urgently needed. The hallmark of HPV cancer is a highly expressed E6/E7 genes of high- risk HPV types. Their expression is necessary for the survival of cervical cancer cell lines. These viral oncogenes are known to dysregulate host DNA damage responses, apoptosis, metabolism, mitotic checkpoint, and immune responses. We propose that inhibitors intervening one or more of these targets in combination with current chemotherapeutic agent, cisplatin, would increase efficacy and reduce morbidity and mortality. NCI encourages drug testing in patient derived cervical tumor xenografts (PDXs) in mice, rather than cancer cell lines. The establishment and inhibitor screening in these model is time consuming, labor intensive and costly, limiting the scope of testing the candidate inhibitors or their combination to predict patient-specific treatment. We have established a novel method to culture cervical cancer as organoid raft cultures as an in vitro tumor model. The organoids can be used to initiate PDX in SCID mice. Conversely, PDX can be grown as organoids. Here, we propose to combine the PDX model with the ease and economy of organoid raft cultures for initial evaluation of new candidate inhibitors. In the first Aim, we propose to validate similarities in phenotypic, genetic, and molecular characteristics among organoids, organoid-derived and patient-derived PDXs in SCID mice and the founder tumors. Significant and precious time and costs will be saved in preclinical investigation of new therapeutic strategies, if the organoid raft culture displays high similarity to the PDX and parental tumors. The HPV E6/E7 oncoproteins dysregulate the cell cycle regulation, the DNA damage and Spindle assembly pathways and metabolism, basis for viral carcinogenesis. The second aim is to evaluate a panel of 6 inhibitors against novel protein targets (Hec1, KIF-11, Aurora A, TTK, HSP90B1, and O-GlcNac Transferase) in the above named pathways. Our preliminary study showed that each was critically required for the survival of cervical cancer cell lines in submerged cultures or HPV transformed cells in vivo. We will also test Vorinostat, a pan HDAC inhibitor, which we found to be promising in our preliminary study. These inhibitors alone or in combination with cisplatin, which is the most commonly used chemotherapeutic agent to treat cervical cancer, will be evaluated in vitro in organoid raft cultures of PDX derived from four independent cervical cancers and in corresponding PDXs in vivo for comparison. The results are expected to validate organoid raft culture as an economical and expedient method of drug evaluation prior to validating in vivo. Additionally we will establish new cc PDXs for future evaluation of agents. These studies will reveal whether the incorporation of these new pathway-specific inhibitors increased the efficacy of current chemotherapy and are candidates for further preclinical investigation.